Cryptococcus neoformans (Cn) causes cryptococcosis, a fungal meningoencephalitis. The pathogen has genetically distinct varieties (var. grubii, var. neoformans, and var. gattii) and mating types (MATalpha and MATalpha). These varieties elicit different host responses in normal and immunocompromised individuals. The mechanisms underlying these pathogenic differences are not well understood. Cn var. grubii is the most common cause of cryptococcosis worldwide; var. neoformans infections have been mostly recognized in Europe. Both primary and opportunistic infections due to var. gattii were till recently reported from Australia, Southeast Asia and South America with rare reports from North America. This epidemiology has changed dramatically with the current report of a major var. gattii outbreak in humans and animals in British Columbia, Canada. Currently, Cn mating is under intensive investigations to define the observed linkage between fungal virulence and MATalpha, and to understand the regulatory signaling pathways. Recently, MATalpha and MATalpha loci from var. grubii and var. neoformans were mapped, and many MAT-specific genes are under intensive study for their role in fungal biology and virulence. However, these powerful molecular genetic analyses have not yet been extended to var. gattii, which will be essential for better understanding of its pathogenesis and the development of more effective therapy. The emerging knowledge from var. grubii and var. neoformans was applied in this laboratory to develop DNA based rapid tests to identify all varieties and mating types; further study of MATalpha/alpha pheromones revealed variety specificity, different copy numbers, and chromosomal locations, and a potential for cross-variety mating and/or hybridization. The current proposal builds upon these investigations by focusing exclusively on var. gattii. Our specific goals are: (i) identification, mapping and phylogenetic studies of genes comprising MATalpha locus, and (ii) characterization of var. gattii STE12alpha cDNA, construction of gene knockout mutants, and study of mutant phenotypes and pathogenic potential. This study may lead to better understanding of var. gattii pathogenicity by providing (a) a genetic map of genes comprising MATalpha locus, (b) characterization of the role of STE12alpha in biology and virulence, and (c) a framework for future genetic investigations of fungal virulence.